Towards Modernization of the Formulation of the Traditional Uighur

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2012, Article ID 863101, 10 pagesdoi:10.1155/2012/863101

Research Article

Towards Modernization of the Formulation of the TraditionalUighur Medicine Herbal Preparation Abnormal Savda Munziq

Murat Kizaibek,1 Ruxandra Popescu,2 Sonja Prinz,2 Halmurat Upur,1

Judith Singhuber,2 Martin Zehl,2 and Brigitte Kopp2

1 Faculty of Traditional Uighur Medicine, Xinjiang Medical University, Urumqi 830011, China2 Department of Pharmacognosy, University of Vienna, Althanstraße 14, 1090 Vienna, Austria

Correspondence should be addressed to Brigitte Kopp, [email protected]

Received 3 January 2011; Revised 6 May 2011; Accepted 20 May 2011

Academic Editor: E. Yesilada

Copyright © 2012 Murat Kizaibek et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Abnormal Savda Munziq (ASMq) is a herbal preparation used in Traditional Uighur Medicine for the treatment and preventionof diabetes, cardiovascular diseases, chronic asthma and cancer. The recommended dose of this decoction for cancer patientsis 500 mL administered orally three times a day. Our approach aimed at reducing the high amount of fluid intake requiredby fractionation of ASMq guided by the antiproliferative activity on HL-60 cells. The fractionation of ASMq resulted inthe preparation of an active extract, Extr-4. Using solid phase extraction, Extr-4 was further fractionated into five fractions(SPE-0, SPE-20, SPE-40, SPE-60 and SPE-80), with SPE-40 showing the strongest antiproliferative activity. Caffeic acid, rutin,isoquercitrin, isorhamnetin 3-O-rutinoside, apigenin 7-O-glucoside, rosmarinic acid, luteolin and formononetin were identifiedin Extr-4 and fractions thereof by means of TLC, HPLC-DAD and LC-MS. SPE-40 contained the main compounds responsiblefor the antiproliferative activity on HL-60 cells. Thus, a phenolic fraction with high antiproliferative activity on HL-60 cells wasobtained from ASMq through the bioassay-guided fractionation process. This could provide a better pharmaceutical formulationthat minimizes the administration inconveniencies of a high volume (1.5 L per day) of ASMq decoction for cancer patients.

1. Introduction

Abnormal Savda Munziq (ASMq) is a medicinal herbalpreparation used in the traditional Uighur medicine inXinjiang region of China [1]. ASMq includes ten medicinalspecies represented by Cordia dichotoma Forst. f., Anchusaitalica Retz., Glycyrrhiza uralensis Fisch., Adiantum capillus-veneris L., Euphorbia humifusa Willd., Ziziphus jujuba Mill.,Lavandula angustifolia Mill., Foeniculum vulgare Mill., Me-lissa officinalis L., and Alhagi pseudoalhagi Desv. The mix-ture is administered as a decoction and is regularly used forthe prevention and treatment of diabetes, cardiovasculardiseases, chronic asthma, and cancer. For liver, stomach, andcolon cancer, the recommended dose of the decoction is500 mL three times a day [2].

Recent studies have shown that ASMq can significantlyinhibit the growth and viability of Hep G2 human hepatomacell line [2–4]. ASMq has also been reported to scavenge freeradicals [1]. In addition, some species included in ASMq

were reported to show in vitro inhibitory activity on differenttumor cell lines. For example, G. uralensis inhibited thegrowth of Hep3B human hepatoma [3] and MCF-7 breastcancer cell line [4]. M. officinalis exerted inhibitory activityagainst continuous cell culture of HEp-2 cells derived fromhuman laryngeal cancer [5], HeLa, and MCF-7 cell lines [6].Z. jujuba decreased the viability of Hep G2 cell line [7].Some of these species also showed in vitro antiproliferativeactivities on HL-60 cell line [8].

Although the effect of ASMq and species included inits composition has been recorded on different cancer cells,the high administration volume of the preparation (1.5 Lper day) is not only inconvenient for the patients, but alsoadds with a high percentage to the daily recommended fluidintake. The European Food Safety Authority recommends adrinking volume of around 1.5 L per day. In the USA, TheFood and Nutrition Board 2004 states a required amount offluid intake (water and beverages) of 3 L per day for adultmen and 2.2 L per day for adult women [9–11].

2 Evidence-Based Complementary and Alternative Medicine

Thus, the consumption of 1.5 L day−1 of ASMq wouldrepresent around 100% of the required fluid intake asconsidered by the European forums and around 50% ofthe American recommended fluid intake. Therefore, reduc-ing the volume of ASMq decoction, while preserving theantiproliferative effect of the mixture, would provide a moreconvenient modern formulation for the treatment of cancerpatients. The present study aimed at obtaining a highly activeantiproliferative fraction of ASMq using bioassay-guidedfractionation and cell viability evaluation.

2. Experimental

2.1. Plant Materials. Pobumuguo (fruits of C. dichotoma),Niushecao (whole plant of A. italica), Gancao (root of G. ura-lensis), Tiexianjue (whole plant of A. capillus-veneris), Diji-ncao (whole plant of E. humifusa), Hongzao (fruits of Z.jujuba), Xunyicao (aerial part of L. angustifolia), Xiaohui-xiang (fruits of F. vulgare), Mifenghua (whole plant of M.officinalis), and Citang (sugar secretion from A. pseudo-alhagi) were purchased from Xinjiang Hospital of TraditionalUighur Medicine, Urumqi, China in September 2007. Theplant material used was unprocessed.

2.2. Chemicals and Reagents. Luteolin, rosmarinic acid,apigenin 7-O-glucoside and isorhamnetin 3-O-rutinosidewere obtained from Extrasynthese (Genay, France), caffeicacid from Sigma-Aldrich (St. Louis, Mo, USA), and for-mononetin, rutin, isoquercitrin and gallic acid from CarlRoth (Karlsruhe, Germany). Acetonitrile (HPLC grade) waspurchased from VWR International (Leuven, Belgium).Glacial acetic acid (purity > 99.8%) and DMSO wereobtained from Carl Roth (Karlsruhe, Germany). Folin-Ciocalteu’s phenol reagent was purchased from Merck(Darmstadt, Germany), HPD-300 macroporous resin fromCang Zhou Bonchem Co., Ltd. (Hebei, China), Polyamideresin from Taizhou Luqiao Biochemical Corporation (Zhe-jiang, China), SPE Mega Bond Elut C-18 cartridges fromVarian (Middelburg, The Netherlands). TLC silica gel 60 F254

plates were obtained from Merck (Darmstadt, Germany). Allother reagents used were of analytical grade.

2.3. Preparation and Fractionation

2.3.1. Preparation of ASMq. ASMq was prepared accordingto a previously reported procedure [12]. The plant materialwas separately ground and mixed in the following ratio:C. dichotoma (10.6), A. italica (10.6), G. uralensis (7.1),A. capillus-veneris (4.9), E. humifusa (4.9), Z. jujuba (4.9),L. angustifolia (4.9), F. vulgare (4.9), M. officinalis (4.9),and A. pseudoalhagi (42.3). The mixture was decocted inboiling water in a ratio of 1 : 10 (w/v) for 3 h. After filtration,the residue was reextracted for 3 h, two times in the samevolume of boiling water. The resulting crude extract wasfiltered, evaporated to dryness under reduced pressure, andpulverized. The obtained powder was used for this study.The yield was 39.9% (w/w) with respect to the total mass ofdry materials.

2.3.2. Preparation of Different Extracts of ASMq. 350 g dryextract of ASMq was dissolved in 1050 mL hot H2O (60◦C),filtered, and cooled to room temperature. The resultingsolution was passed through a column (125 cm× 5 cm) filledwith HPD-300 macroporous adsorbent resin. The columnwas eluted with 3 bed volumes (BV) of H2O dest. The H2Oeluate was concentrated, and then 95% EtOH was added toobtain a final concentration of 70% EtOH. The mixture wasstirred for 10 min. The resulting precipitate was collected andredissolved in H2O and the EtOH precipitation was repeatedonce again. The resulting precipitate was decolored withactivated carbon and evaporated under reduced pressure toobtain extract 1 (Extr-1, 54.9 g). The HPD-300 macroporouscolumn was sequentially eluted with 3 BV of 20% EtOH and3 BV of 60% EtOH, and the resulting eluates were collectedseparately and evaporated under reduced pressure to drynessto obtain extract 2 (Extr-2, 15.7 g) and extract 3 (Extr-3,25.3 g), respectively. The yields (w/w) of Extr-1, Extr-2, andExtr-3 were 15.69%, 4.49%, and 7.23% of the dry weight ofASMq, respectively.

2.3.3. Preparation of Extract 4. Dry extract of ASMq (350 g)was dissolved in 1050 mL hot H2O (60◦C) and filtered.1.5 volumes of 95% EtOH were added; the mixture wasstirred for 20 min and then allowed to stand for 24 h.The resulting supernatant layer was filtered, concentratedunder reduced pressure to 350 mL and subjected to columnchromatography (125 cm × 5 cm) on polyamide resin. Thecolumn was eluted first with H2O dest. (3 BV), followedby 60% EtOH (3 BV). The fraction eluted with H2O dest.was discarded, while the fraction eluted with 60% EtOHwas evaporated under reduced pressure to dryness to obtainextract 4 (Extr-4, 12.2 g). The yield (w/w) of Extr-4 was3.49% with respect to the dry weight of ASMq.

2.3.4. Fractionation of Extr-4. Extr-4 was further fractionatedby solid phase extraction (SPE). 50 mg of Extr-4 weredissolved in 1 mL MeOH 40% and applied onto an SPEcolumn preconditioned with 2 reservoir volumes (RV)MeOH and 2 RV H2O. The cartridge was sequentially elutedwith 2 RV of H2O and 20%, 40%, 60%, and 80% MeOH at aflow rate of 1 mL min−1. The resulting eluates were collectedseparately and evaporated under reduced pressure to yieldSPE-0 (1.1 mg), SPE-20 (1.0 mg), SPE-40 (11.7 mg), SPE-60 (11.9 mg) and SPE-80 (5.2 mg), respectively. The yields(w/w) of these fractions were 0.15% (SPE-0), 0.14% (SPE-20), 1.63% (SPE-40), 1.66% (SPE-60), and 0.73% (SPE-80)of the dry weight of ASMq, respectively.

A flow chart of the ASMq preparation and fractionationprocedure is shown in Figure 1.

2.4. Cell Culture. The HL-60 human promyelocytic leukemiacell line was obtained from the American Type CultureCollection (ATCC). Cell medium RPMI 1640 and its supple-ments were obtained from Life Technologies, Inc., USA. TheHL-60 cells were routinely cultured in RPMI 1640 medium,supplemented with 10% (v/v) heat inactivated foetal bovine

Evidence-Based Complementary and Alternative Medicine 3

Dry mixture of ten herbs

ASMq

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Figure 1: A flow chart of the ASMq preparation and fractionation procedure.

serum, 1% L-glutamine, and 1% penicillin/streptomycin andgrown in a humidified atmosphere with 5% CO2 at 37◦C.

2.5. Evaluation of Cell Viability. Cells were seeded at adensity of 0.1 × 106 cells mL−1 in 12-well plates and grownfor 24 h. Cells were then incubated with 100 μg mL−1 and250 μg mL−1 ASMq, extracts (Extr-1, Extr-2, Extr-3, andExtr-4) and 50 μg mL−1 SPE fractions (SPE-0, SPE-20, SPE-40, SPE-60, and SPE-80), respectively. In order to determinethe IC50 value of Extr-4, cells were exposed to increasingconcentrations (0, 25, 50, 100, 200, and 250 μg mL−1) of Extr-4. Prior to use, the stock solutions of all dry extracts (ASMqextracts and SPE fractions) were prepared in 60% EtOHand then filtered through 0.20 μm sterile syringe filters. Thefinal concentration of EtOH in culture medium during thetreatment of cells did not exceed 0.5% (v/v). Vehicle-treatedcells (0.5% EtOH) were tested as control samples. After 24 h,48 h, and 72 h of incubation, the cells were stained withtryptan blue (0.4%) and counted with a cell counter (Vi-CELL XR Cell Viability Analyzer, Beckman, USA). The cellviability was expressed as the percentage of control. Analyseswere performed in triplicate, values are given as mean ± SD.

2.6. TLC Analysis. TLC analyses of ASMq extracts and SPEfractions were performed on silica gel 60 F254 plates. Extr-1,Extr-2, Extr-3, and Extr-4 were dissolved in H2O, 20% EtOH,40% EtOH, and 60% EtOH, respectively (20 mg mL−1), and10 μL aliquots were applied to a TLC plate. As mobile phaseCHCl3/MeOH/H2O (70 : 22 : 3.5, v/v/v) was used, the platewas sprayed with 0.5% fast blue salt B aqueous solution [13].Evaluation was done under visible light. Rosmarinic acid wasused as reference compound.

SPE fractions were dissolved in 60% MeOH (20 mgmL−1) and 5 μL aliquots were applied to a TLC plate anddeveloped with EtOAc/HCOOH/HAc/H2O (100 : 11 : 11 : 26,v/v/v/v). The plate was then sprayed with natural productsspraying reagent (1% methanolic diphenylboric acid-β-ethylamino ester) followed by 5% ethanolic polyethyleneglycol-4000 [13]. The plate was investigated under 365 nm.Rutin, hyperoside, and astragalin were used as referencecompounds.

2.7. HPLC-DAD and LC-MS Analysis. HPLC-DAD analysewas conducted on a Shimadzu LC-10AD liquid chromato-graph equipped with a SCL-10A system operator, a SPD-M20A diode array detector, a SIL-10AD auto injector and aDGU-14A degasser. The LC-MS analyses were performed onan UltiMate 3000 RSLC-series system (Dionex, Germering,Germany) coupled to a 3D quadrupole ion trap massspectrometer equipped with an orthogonal ESI source (HCT,Bruker Daltonics, Bremen, Germany). HPLC separation wascarried out on a Hypersil BDS-C18 column (4 × 250 mm,5 μm, Thermo Scientific, Waltham, Mass, USA) at a flow rateof 1.2 mL min−1. Water (pH 2.8 with acetic acid) and MeCN(with the same amount of acetic acid) were used as mobilephase A and B, respectively. The following gradient programwas used: 15% B (0 min), 41.3% B (45 min), 95% B (46 min),and 95% B (51 min). The eluent flow was split roughly 1 : 8before the ESI ion source, which was operated as follows:capillary voltage: 3.7 kV, nebulizer: 30 psi (N2), dry gas flow:8 L min−1 (N2), and dry temperature: 340◦C. The massspectrometer was operated in an automated data-dependentacquisition (DDA) mode where each MS scan (m/z 80–1100, average of 5 spectra) was followed by MS2 scans (m/z40–1100, average of 3 spectra, isolation window of 4 Th,


fragmentation amplitude of 1.0 V) of the two most intenseprecursor ions, and MS3 scans (m/z 40–1100, average of 3spectra, isolation window of 4 Th, fragmentation amplitudeof 1.0 V) of the most intense fragment ion in each MS2

scan. In additional LC-MS experiments, the instrument wasoperated in alternating ion MS1 mode.

The sample injection volume was 10 μL. 2 mg of Extr-4as well as 2 mg of each of the SPE fractions were separatelysonicated with 1 mL of 60% MeOH-DMSO (4 : 1, v/v) for5 min at room temperature. Standard phenolic compoundswere dissolved in MeOH. Identification of compounds wasachieved by comparing retention times as well as UV andMSn spectra.

2.8. Total Flavonoid Content. The total flavonoid content wasdetermined by aluminium nitrate method [14]. 30 mg ofASMq and 3 mg of Extr-4 were dissolved in 1 mL H2O and1 mL of 60% EtOH in a 5 mL volumetric flask, respectively.2 mL of H2O and 0.15 mL of aqueous 5% NaNO2 solutionwere added, followed by 0.15 mL of 10% Al(NO3)3 6 minlater. The mixture was allowed to stand for another 6 minat room temperature. Then, 1 mL of 1 M NaOH was added.The sample was immediately diluted with H2O to a finalvolume of 5 mL and mixed. A blank without sample solutionwas prepared in parallel. Rutin was used as standard toestablish the calibration curve. Absorbances of samples weredetermined at 510 nm on a spectrophotometer (BeckmanDU-640). Total flavonoid content was expressed as mg rutinequivalent (RUE) g−1 dried extract. Data for ASMq andExtr-4 were reported as mean ± SD for five replicates andfor triplicates, respectively.

2.9. Total Phenolic Content. The Folin-Ciocalteu method wasused to determine the total phenolic content [15]. 160 μLof sample (6 mg ASMq dissolved in 1 mL H2O and 0.6 mgExtr-4 dissolved in 1 mL 60% EtOH, respectively) were addedto a 5 mL volumetric flask and diluted with H2O dest.to 2.5 mL. 0.25 mL of Folin-Ciocalteu reagent were added.6 min later, 0.75 mL of 20% aqueous Na2CO3 solution wereadded, diluted with H2O to a final volume of 5 mL andmixed. After incubation at room temperature for 2 h, theabsorbance of the reaction mixture was measured at 765 nmagainst a blank containing only solvent and reagents. Gallicacid was used as standard for the calibration curve. Totalphenolic content was expressed as mg gallic acid equivalent(GAE) g−1 dried extract. Data for ASMq and Extr-4 werereported as a mean± SD for five replicates and for triplicates,respectively.

2.10. Statistical Analysis. The results of cell viability were pre-sented as means± SD. Statistical comparison was performedusing the one-way analysis of variance (ANOVA) followed byTukey’s test (in case of equal variance) or Dunnett’s T3 test(in case of unequal variance). These tests were performedusing SPSS 13 for Windows. P < 0.05 was consideredstatistically significant. IC50 values and their 95% confidencelimits were estimated by a sigmoidal dose-response modelwith variable slope (GraphPad Prism software, version 4.03).

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Figure 2: Effect of ASMq, Extr-1, Extr-2, Extr-3, and Extr-4 on theviability of HL-60 cells. Cells were seeded in 12-well plates, grownfor 24 h and then treated with 100 μg/mL and 250 μg/mL ASMq,Extr-1, Extr-2, Extr-3, and Extr-4. After 24 h, 48 h, and 72 h cellviability was assessed by tryptan blue exclusion method. Data rep-resent the percentage of control value and are expressed as mean ±SD of triplicate cultures. ∗P < 0.05, as compared to control cells.

3. Results and Discussion

In the first step of this study, three extracts, Extr-1, Extr-2, and Extr-3, were obtained from ASMq by macroporousresin chromatography and tested for their effect on cellviability. Figure 2 shows the activity of ASMq and thecorresponding extracts on HL-60 cell viability. ASMq andExtr-1 (250 μg mL−1) showed no remarkable cytotoxicityafter 72 h, reducing cell viability to 87.1 ± 16.7% and 87.7 ±1.3%, respectively. Extr-1 enhanced cell proliferation after24 h for both tested concentrations (100 and 250 μg mL−1).ASMq was previously reported to contain 58% saccharidesas glucose equivalents [16]. Therefore, the enhanced cellproliferation of HL-60 cells after 24 h in the presence of Extr-1 could be due to the high content of saccharides in theseextracts. Unlike ASMq and Extr-1, Extr-2 (250 μg mL−1)showed higher cytotoxicity, inducing a decline in cell viabilityto 64.8 ± 4.8%, while Ext-3 (250 μg mL−1) exhibited thehighest cytotoxicity, decreasing cell viability to 50.83 ±0.70% after 72 h. Thus, ASMq showed no remarkable activityon HL-60 cell proliferation at the given concentration, whiledifferent effects on cell viability were distributed between thethree extracts obtained from ASMq, with the most apolar ofthe extracts showing increased antiproliferative effect. Thelow activity of ASMq is in line with the high dose of ASMqdecoction (1.5 L per day) required for the treatment of cancerpatients. The result of TLC analysis revealed that phenoliccompounds were absent in Extr-1 but could be detectedin Ext-2 and Extr-3. When compared to Extr-2, Extr-3showed an enriched phenolic fingerprint. This implied apossible correlation between the phenolic compounds andthe cytotoxic activity on HL-60 cells. Therefore, a polyamidechromatography was performed on ASMq to obtain anextract enriched in phenolic compounds. The resulting


extract, Extr-4, showed higher cytotoxicity than Extr-2 andExtr-3, reducing cell viability to 5.29± 0.28% at a concentra-tion of 250 μg mL−1, after 72 h (Figure 2). Figure 3 shows thedose-response curve for Extr-4 when tested on HL-60 cellsafter 24 h, 48 h, and 72 h. The IC50 values were 105.7 μg mL−1

(24 h), 95.1 μg mL−1 (48 h) and 72.3 μg mL−1 (72 h).According to preliminary TLC analysis, the number

of phenolic compounds increased from Extr-1 to Extr-4with the majority present in the latter. The total flavonoidcontent and total phenolic content of ASMq were 25 ±1 mg RUE g−1 dried extract and 38 ± 1 mg GAE g−1 driedextract, respectively, whereas those of Extr-4 were 221 ±9 mg RUE g−1 dried extract and 333 ± 7 mg GAE g−1 driedextract, respectively. Both the flavonoid content and thephenolic content increased to approximately 9-fold in Extr-4versus ASMq. These results confirmed that the phenoliccompounds were enriched in Extr-4 through polyamide resinchromatography.

Using SPE, Extr-4 was further fractionated into fivefractions, SPE-0, SPE-20, SPE-40, SPE-60, and SPE-80.According to cell viability assessment after 72 h, all fractionsshowed various degrees of cytotoxicity on HL-60 cells(Figure 4). SPE-40 (50 μg mL−1) was found to exhibit thestrongest inhibitory effect on the viability of HL-60 cells byinducing a time-dependent decrease in cell viability to 8.2 ±1.5%. The TLC analyses revealed the presence of flavonoidsin all SPE fractions except SPE-80. The majority of flavonoidswere present in SPE-40, implying that flavonoids may be themost active phenolic compounds in SPE-40.

The identification of phenolic compounds in Extr-4 andSPE fractions was performed using HPLC-DAD and LC-MS. The HPLC chromatogram of Extr-4 recorded at 254 nmis shown in Figure 5. Eight phenolic compounds, namely,caffeic acid (1), rutin (2), isoquercitrin (3), isorhamnetin3-O-rutinoside (4), apigenin 7-O-glucoside (5), rosmarinicacid (6), luteolin (7), and formononetin (8), were identifiedin Extr-4 by comparison of their retention time, and UVand MS spectral characteristics to those of standards inFigure 5(a), as well as by spiking the sample with standards.The chemical structures of compounds 1∼8 are shownin Figure 6. Several phenolic compounds that have beendescribed as chemical constituents of the species included inASMq, such as chlorogenic acid, luteolin 3′,7-O-diglucoside,ferulic acid, eriodictyol 7-O-glucoside, narirutin, rhamnetin,rhoifolin, and isovitexin, were not found in Extr-4. Some ofthe phenolic compounds we identified in Extr-4 by LC-MSwere also found in SPE-0, SPE-20, SPE-40, and SPE-60. Forexample, 1, 2, and 6 were identified in SPE-0 (Figure 7(a))and SPE-20 (Figure 7(b)) in varying ratios. Five phenoliccompounds, 2–6, were identified in SPE-40 (Figure 7(c)),whereas 4, 7, and 8 were found in SPE-60 (Figure 7(d)).Compound 3 was identified only in SPE-40. Based on theresults of TLC and HPLC analyses, SPE-80 may not containphenolic compounds (Figure 7(e)) but is mainly enriched inlicorice triterpene saponins (e.g., glycyrrhizic acid) accordingto tentative identification by LC-MS (data not shown).

Phenolic compounds are well studied secondary plantmetabolites that have been thoroughly investigated dueto their presence in the human diet and their biological

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Figure 3: Dose-response curves of antiproliferative activity ofExtr-4 after 24 h (a), 48 h (b), and 72 h (c). HL-60 cells werecultured in 12-well plates for 24 h to reach logarithmic growthphase and then incubated with 25–250 μg/mL Extr-4. Cell viabilitywas estimated by tryptan blue staining and cell counting after24 h, 48 h, and 72 h. Data are presented as percentage of controlvalue. The horizontal axis represents a log scale of concentrationof Extr-4. The IC50 was calculated using a sigmoidal dose-responsemodel with variable slope. The curve represents the average ofexperiments performed in triplicate. ∗P < 0.05, compared withrespective control value (24 h : 100.00 ± 8.50%; 48 h : 100.00 ±8.84%, 72 h : 100.00 ± 22.26%).


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Figure 4: Viability of HL-60 cells after 24 h, 48 h, and 72 hof treatment with SPE-0, SPE-20, SPE-40, SPE-60, and SPE-80subfractions. Cell viability was assessed by tryptan blue exclusionmethod and is presented as percentage of control value. Data areexpressed as mean ± SD of two independent experiments, each ofwhich was done in triplicate. ∗P < 0.05, compared with control.Each sample was tested at a concentration of 50 μg/mL.

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Figure 5: Reversed phase HPLC chromatograms of standardphenolic compounds (a) and Extr-4 (b) at a wavelength of 254 nm.(1) caffeic acid, (2) rutin, (3) isoquercitrin, (4) isorhamnetin 3-O-rutinoside, (5) apigenin 7-O-glucoside, (6) rosmarinic acid, (7)luteolin, and (8) formononetin.

properties, including their beneficial effects on health. Oneof these effects is their capacity to interfere with all stagesof the cancer process [17, 18]. Several flavonoids have beenshown to suppress carcinogenesis in various animal models[19]. A growing number of epidemiological studies suggestthat high flavonoid intake may be correlated with decreasedcancer risk [20–22].

The phenolic compounds detected in Extr-4 and its SPEfractions were also previously identified in species from thecomposition of ASMq. For example, 1 was identified in L.angustifolia [23] and M. officinalis [24], 2 in Z. jujuba [25],A. capillus-veneris [26], C. dichotoma [27], G. uralensis [28],and F. vulgare [29], and 3 in A. capillus-veneris [26], G.uralensis [28], and M. officinalis [30]. 4 was found in C.dichotoma [27], G. uralensis [28], and F. vulgare [29] and 5

in E. humifusa [31] and M. officinalis [30]. 6 was identified inC. dichotoma [27], F. vulgare [29], and M. officinalis [24]. 7and 8 were found in M. officinalis [32] and G. uralensis [33],respectively.

Compounds 2 [34] and 8 [35] were reported to possessno remarkable cytotoxicity on HL-60 cells. 4 showed nodistinctive cytotoxicity on HT-29 human colon carcinoma,MCF-7 human breast carcinoma, and Hep G2 humanhepatoma cell lines [36]. 3 showed antiproliferative effects onB16F10 murine melanoma [37] and MCF-7 human breastcarcinoma cell lines [38]. 5 possessed moderate cytotoxiceffects on human prostate cancer cell lines derived fromdifferent metastatic sites, such as LNCaP, DU145, and PC-3 cells [39]. 1 [40] and 6 [41] showed low cytotoxic effectson HL-60 cell line, while 7 [42] exhibited a strong cytotoxicactivity on HL-60 cells. Thus, the phenolic compoundsidentified in Extr-4 have been previously shown to have noremarkable (2, 4, 8), moderate (1, 3, 5, 6), or strong (7)cytotoxic effect on different cancer cell lines.

SPE-0 and SPE-20 contain compounds that have beendescribed as moderate cytotoxic which can explain theirobserved activity in the viability tests. The concentrationof 7, a strong cytotoxic compound, in SPE-60 was lowas shown by the HPLC analysis, justifying the moderateeffect of this fraction. The characterization of SPE-40, themost active fraction, identified compounds described asmoderate (3, 5, 6) or very low cytotoxic (2, 4), suggestingthat 3, 5, and 6 could, at least in part, contribute to theantiproliferative activity of SPE-40 on HL-60 cells. Themechanisms of antiproliferative effects of these compoundswere investigated in some studies. For example, 3 wasreported to reduce glioblastoma cell growth without induc-ing apoptosis, possibly by modulating the control of thecell cycle. It was also suggested that β-catenin-mediatedsignaling may be involved on the antiproliferative activity of3 [43]. 5 inhibited cancer cell growth through deconjugationof glycosides that occurs in the cellular compartment toproduce aglycone and apigenin [44]. Apigenin itself exhibitsa significant growth inhibition against hepatoma cell linesand induces apoptosis in Hep G2 cells [45]. Its apoptoticmechanism might be mediated through the p53-dependentpathway and the induction of p21 expression [45] or througha mitochondria/caspase-pathway [46]. It was also reportedthat apigenin inhibits cancer cell proliferation through G2/Mcell cycle arrest [47] and Erβ [48]. The authors have found noreport on the mechanism underlining the antiproliferativeactivity of 6 against cancer cells.

However, these results also imply that the identifiedphenolics are not the only compounds responsible forthe antiproliferative activity of SPE-40 and suggest thepossible presence of one or more compounds with strongeractivity. The latter could contribute to the cytotoxicity ofthe identified phenolic compounds by both additive andsynergistic effects. The majority of the uncharacterized com-pounds in SPE-40 showed UV-Vis spectral characteristicssimilar to flavanones, chalcones, and ellagic acid derivatives(data not shown), which were reported to possess strongantiproliferative activities on different cancer cells [49–52].This could also be confirmed by LC-MS, which indicates the


COOH

OH

OH

OH

OH

OH

OH

OH

OHHO

HO

HO

HO

HO HO

O

O

O O

OO

OO

O

O

R1

R2O

R3

CH3

1

6

7 8

2

3

4

5

R1 = –O–Rutinose, R2 = H, R3 = OH

R1 = –O–Glc, R2 = H, R3 = OH

R1 = –O–Rutinose, R2 = H, R3 = OCH3

R1 = H, R2 = –O–Glc, R3 = H

Figure 6: Compounds identified in Extr-4 and its fractions: (1) caffeic acid, (2) rutin, (3) isoquercitrin, (4) isorhamnetin 3-O-rutinoside,(5) apigenin 7-O-glucoside, (6) rosmarinic acid, (7) luteolin, and (8) formononetin.

presence of several typical liquorice flavanones (e.g., liquir-itin apioside, liquiritin, and liquiritigenin) and chalcones(e.g., isoliquiritin apioside and isoliquiritin) based on thecomparison of multistage MS data with published results(data not shown) [53], indicating that phenolic compounds,with possible strong antiproliferative activity, constitute mostof the remaining uncharacterized compounds in SPE-40.

4. Conclusion

In summary, our study focused on the bioassay-guidedfractionation of ASMq aiming at the reduction of theadministration volume of the ASMq decoction (1.5 L day−1)and the modernization of its pharmaceutical formulation.

Four extracts, namely, Extr-1, Extr-2, Extr-3, and Extr-4,were prepared from ASMq. Extr-4 exerted the strongestantiprolifertive activity on HL-60 cells. Extr-4 was furtherfractionated into five fractions by solid phase extraction, ofwhich SPE-40 showed the strongest inhibitory effect on HL-60 cells. The results of the cell viability assay, quantificationassay, and the TLC and LC-MS analyses on Extr-4 and theSPE fractions revealed that the main bioactive componentswith antiproliferative activity against HL-60 cell line couldbe phenolic compounds. The substances identified in Extr-4and its SPE fractions were caffeic acid, rutin, isoquercitrin,isorhamnetin 3-O-rutinoside, apigenin 7-O-glucoside, ros-marinic acid, luteolin, and formononetin. Isoquercitrin,apigenin 7-O-glucoside, and rosmarinic acid may contribute


02.

5 57.

5 1012

.5 1517

.5 2022

.5 2527

.5 3032

.5 3537

.5 4042

.5

−500

50100150200250

0255075

100125

0

20

40

60

800

50

100

150

2000

100

200

300

400

500

1

1 2

2

2

3

4

4

5

6

6

6

7

8

(a)

(b)

(c)

(d)

(e)

(min)

(mA

U)

Figure 7: HPLC chromatograms of five SPE fractions at a wave-length of 254 nm. (a) SPE-0, (b) SPE-20, (c) SPE-40, (d) SPE-60, (e) SPE-80, (1) caffeic acid, (2) rutin, (3) isoquercitrin, (4)isorhamnetin 3-O-rutinoside, (5) apigenin 7-O-glucoside, (6)rosmarinic acid, (7) luteolin, and (8) formononetin.

to the cytotoxicity of SPE-40 on HL-60 cells. The results alsosuggest that other compounds than the identified phenolicsadd to the antiproliferative effect of ASMq. However, fractionSPE-40 obtained in the course of our work could significantlyreduce the high therapeutic dosage of ASMq (500 mL threetimes a day). One dose of ASMq was reduced from 500 mL(∼200 g dry weight) to 1.6 g fraction SPE-40. Thus, thebioassay-guided fractionation of ASMq could lead to theenrichment of active compounds in an active fraction andto the diminution of the administration dose of the TCMpreparation, providing a better pharmaceutical formulationfor cancer patients.

Acknowledgments

The first author is thankful to Eurasia Pacific Uninet forproviding the scholarship. This research work is part of theproject “Quality Assurance of Herbal Medicinal Products(HMPs) from TCM”, aided by the Austrian Federal Ministryof Science and Research and the Federal Ministry for Health,Family, and Youth and was also funded by the CultivationFund of the Key Scientific and Technical Innovation ProjectMinistry of Education, China (no. 708058). The authors wish

to present their acknowledgements also to Katharina Tscher-now for her contribution to the fractionation of Extr-4.

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